Multi-omics Analysis of S. Pneumoniae Extracellular Vesicles

Extracellular vesicles (EVs) are cell-derived nanoparticles that can serve as carriers of DNA, RNA, and protein. For the pathogen S. pneumoniae,which causes diseases such as pneumonia and meningitis, EVs can be used to transport these macromolecules to recipient cells, facilitating communication and signaling between cells. However, the overall composition of these S. pneumoniaeEVs is still unknown and how the macromolecules packaged within these EVs contribute to the pathogenicity of S. pneumoniaeremains a hot research topic. In this work, we have developed a novel multi-omic workflow that isolates DNA, RNA and protein from a single S. pneumoniaeEV starting sample, enabling analysis of these macromolecules without the need for multiple sample preparation steps and allowing us to gain insights into the composition and functionality of S. pneumoniaeEVs. EVs from a S. pneumoniaeculture were isolated and the proteins in the EV homogenate were labeled with the bifunctional ProMTag. One end of the ProMTag forms a reversible, covalent bond with proteins. ProMTag's other functional group is methyltetrazine, which forms an irreversible bond with trans-Cyclooctene (TCO), allowing capture of EV proteins on TCO-agarose beads. Organic solvent was then added to precipitate the nucleic acids in the EV homogenate. Nucleic acids were released by a series of washes and subsequently separated into DNA and RNA fractions by RNase or DNase treatment, respectively. Proteins were then released from the TCO-agarose beads by reversing the ProMTag-protein linkage, yielding proteins in their original, unmodified state ready for analysis. DNA and RNA were sequenced, and proteins were identified using mass spectrometry. Using this workflow, we were able to describe the composition of multiple macromolecules within S. pneumoniaeEVs and gain insights into how these macromolecules might facilitate communication and infection within the S. pneumoniaepopulation. © FASEB.